Nascent Polypeptide Chains Exit the Ribosome in the Same Relative In

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Nascent Polypeptide Chains Exit the Ribosome in the Same Relative Position in Both Eucaryotes and Procaryotes

CARMELO BERNABEU,*[I ELAINE M. TOBIN,*II AUDREE FOWLER,*{} IRVING ZABIN,*§ and JAMES A. LAKE*l[ * Molecular Biology Institute and II Department of Biology, University of California, Los Angeles, California 90024; and § Department of Biological Chemistry, University of California Medical School, Los Angeles, California 90024. Dr. Bernabeu's present address is Sidney Father Cancer Institute, Boston Massachusetts 02115.

ABSTRACT We located the polypeptide nascent chain as it leaves cytoplasmic ribosomes from the plant Lemna gibba by immune electron microscopy using antibodies against the small subunit of the enzyme ribulose-l,5-bisphosphate carboxylase. Similar studies with Escherichia coil ribosomes, using antibodies directed against the enzyme //-galactosidase, show that the polypeptide nascent chain emerges in the same relative position in plants and bacteria. The eucaryotic ribosomal exit site is on the large subunit, ~75 ,g, from the interface between subunits and nearly 160 ,~ from the central protuberance, the presumed site for peptidyl transfer. This is the first functional site on both the eucaryotic and procaryotic ribosomes to be determined.

It has been appreciated for some time that, in general, eucar- sites on the large subunits of both ribosomal types. This site is yotic and procaryotic ribosomes share common structural fea- 150 A from the presumed site of the peptidyl-transferase (11). tures (for reviews, see references 1, 2). With the advent of In the duckweed, Lemna gibba, the polypeptide nascent chain immune electron microscopy (3, 4), however, the Escherichia emerges from the large subunit at a region on the side of the coli ribosome has become better understood than its eucaryotic ribosome opposite the translational domain and in the same counterparts. In E. coli, the functional sites involved in trans- relative position as found in the E. coil ribosome. Hence, in lation are clustered into part of the ribosome, comprising spite of the greater complexity of eucaryotic as compared to approximately two-thirds of its volume, referred to as the procaryotic ribosomes, the overall organization of the exit "translational domain." Functional sites contained in the trans- domain on ribosomes, as reflected by the location mapped for lational domain include the initiation factor binding sites (5, the exit site, seems to be similar in both. 6), the messenger binding site (7-9), the peptidyl transferase (10-12), the 5S RNA (13), and the L7/L12 proteins (14) that MATERIALS AND METHODS mediate the GTP-dependent steps of translation (for a review Preparation of Polysomes from E. coil: 2.5 x 10it cellsfrom E. of these locations, see reference 15). Together, these sites define cell A324-5 (17) were resuspended in 2 ml of buffer A (150 mM NH~C1/20 mM the translational domain. Corresponding structural regions are Tris.HCl, pH 7.6/10 raM MgCh) and disrupted in a French pressure ceU at found in eucaryotic ribosomes, as well as in archaebacterial 13,800 psi. The ceU extract was centrifuged in a SS-34 rotor for 8 nun at 8,000 rpm. The supernatant containing the polysomes was layered (0.6 ml per tube) on ribosomes (16). top of a 15-30% sucrose gradient with 0.5 ml of 60% sucrose cushion on the Other aspects of ribosomal organization, particularly those bottom in buffer B (150 mM NFLC1/20 mM Tris.HCl, pH 7.6/5 mM MgCh). involved with protein secretion and processing, could possibly The polysomes were pelleted at 45,000 rpm for 125 min in a SW50.1 rotor differ extensively in eucaryotic and procaryotic ribosomes since (Beckman Instruments, Inc., Spinco Div., Palo Alto, CA). The superuatant was discarded and the pellet rinsed immediately with 5 ml of cold buffer B in order the rough endoplasmic reticulum has no obvious counterpart to eliminate the remaining sucrose. The polysomes were resuspended in buffer B in the procaryotic cell. Here we report investigations on the (400/~1) and stored at -80°C. The yield of polysomes was A~o units per 1011 location of the polypeptide nascent chain as it exits from the cells. ribosome in both procaryotes and eucaryotes. We have mapped Preparation of Polysomes from L. gibba: L. gibba plantsG-3 the exit site of the nascent chain on ribosomes synthesizing the (18) in growth medium were washed with distilled water at room temperature enzymes fl-galactosidase and the small subunit of ribulose-1,5- and poured onto liquid nitrogen or crushed ice at -20°C. -35 g of/.,, gibba were macerated in a mortar and the paste resuspended in 70 ml of extraction buffer bisphosphate carboxylase (Rubisco) using antibodies directed (17% sucrose/0.4 M KC1/30 mM MgCIz/50 mM Tris, pH 9.0). AU the operations against these proteins. were carried out at 4°C. The mixture was ground in a Waling Blender (Waling The exit sites are at a single region located at comparable Productioas Div., Dynamics Corp. of America, New Hartford, CT) four times

THE JOURNAL Of C£LL 81OtOGV - VOI.UM[ 96 MAY 1983 1471-1474 © The Rockefeller University Press - 0021-9525/83/05/1471/04 $1.00 1471 for 15 s, with 1-min intervals. The homogenate was poured through two layers and then eight layers of cold miracloth. The solution was spun for 7 rain at 3,000 A B rpm in a SS-34 rotor. The volume of the supematant was recorded and 0A vol of 70S 20% Triton X-100 was added. The mixture was spun for 20 rain at 12,000 rpm in a SS-34 rotor. The supematant was layered, with a wide pipette, on top of 5 ml of sucrose cushion (60% sucrose/0.2 M KC1/20 raM Tris[pH 7.6]/5 mM MgC12), and more extraction buffer was added to equilibrate all the tubes. After centrifugation for 3 h at 49,000 rpm in a 50.2 Ti rotor (Beckman Instruments, Inc.), the supernatant was discarded and the pellet washed with resuspension buffer (50 mM KCI/20 mM Tris[pH 7.6]/5 mM MgC12). The pellet was finally resuspended in 200/.d of resuspension buffer. These polysomes can be further purified by centrifugation in a SW 50.1 rotor on a sucrose gradient (0.5 ml of 60% sucrose TOP BOTTOM TOP BOTTOM cushion at the bottom and 15-30% sucrose gradient in buffer C [@2 M KCt, 20 mM Tris and 5 mM MgC12]) by spinning at 45,000 rpm for 2 h. FIGURE 1 Analysisof polysomes. (A) L.gibba. (B) E. coil Polysomes (2.1 A2eo U) were layered onto a 15-30% sucrose gradient in buffer Purification of tbe Proteins: The enzyme ribulose-1,5-bisphosphate carboxylase was purified from L. gibba G-3 (18). 20 g of L. gibba were mixed B ( £. gibba) or A ( E. coil) and centrifuged (5W50.1 rotor) for 30 rain. with 10 ml of buffer D (100 mM KC1, 20 mM Tris-HCl, pH 7.4, and 5 mM at 245,000 g. MgCI2), 6 g ofDowex 1 (I × 200--400) and 20 g of sand. The mixture was ground in a mortar and the suspension of the homogenized plant was fdtered through two and then eight muslin layers. The fdtrate was centrifuged for 20 rain at 10,000 rpm in a SS-34 rotor. The clear superuatant was then centrifuged for 30 70S B rain at 45,000 rpm in a SWS0.1 rotor. The supematant was concentrated by either polyethylene glycol (PEG) in a dialys/s bag or by nitrafdtration in an Amicon apparatus (Amicon Corp., Scientific Sys. Die., Danvers, MA). When the volume was ~5-8 ml, the solution was dialyzed overnight against 100 mM KCI, 20 mM Tris-HCl, pH 7.4, and 5 mM MgCI2. The dialysate (1-2 ml Per tube) was layered on top of a 5-30% sucrose gradient in 25 mM Tris-HC1, pH 7.4, and 15 mM MgClz and centrifuged in a VTi 50 rotor (Beckman Instruments, Inc.) for 160 rain at 49,000 rpm. Under these conditions the ribulose-1,5-bisphosphate carboxylase enzyme complex (Mr ~500,000) moves into the gradient ahead of the bulk of other proteins. The Peaks of the enzyme were pooled and dialyzed for 48 h TOP BOTTOM TOP BOTTOM against 2 L of 5% acetic acid (with one change). The acidic solution was then lyophilized. The subunits of the enzyme were separated by preparative SDS FIGURE 2 Isolation of dimers linked by IgG's (A) L. gibba. (B) E. electsophoresis of the lyophilized protein. The small (13,000) and large (52,000) co#. 4 A2eo U of polysomes were reacted with IgG and digested with subunits were localized on the gel by cutting and staining with Coomassie Blue RNase A as described in Materials and Methods. The final mixture a l-cm strip on one side of the slab gel. The unstained gel strips of large and was layered on top of a 15-30% sucrose gradient in buffer 13 (l. small subunits were soaked in water for 3 h in order to remove the SDS. The gels gibba) or A (E. coil) and centrifuged (VTi 65 rotor) for 35 min at were then lyophillzed for 48 h and ground to a free powder in a mortar. The 113,000 g. The shaded dimer peak was collected and negatively enzyme fl-galactosidase was purified as previously described (17). stained With 1% uranyl acetate. Preparation of A n tibodies: To prepare antibodies against the small and large subunits of ribulose-l,5-bisphosphate carboxylase, 1 ml of 0.15 M NaC1, and 0.1 M phosphate buffer, pH 7.0, was added to a gel powder of each previously documented (18, 19). After the formation of intra- protein and mixed with 1 ml of complete Freund's adjuvant. These mixtures were subcutaneously injected on the back of rabbits with a large needle. This was polysomal IgG dimers, the mixture was treated with RNase in followed by an intramuscular boost in the thigh with incomplete Freund's order to cleave the messenger RNA. Dimers of monosomes adjuvant. Preparation of antibodies against the enzyme ~8-galactosidase has been linked by IgG's were then separated from other components described (19). Purification of the IgG fractions was done by passing the rabbit on a sucrose gradient. Fig. 2 shows the resolution obtained serum through a Protein A-Sepharose 4B column (Pharmacia Fine Chemicals, with this separation. The shadowed areas contain the IgG Piscataway, N J) (20). Preparation of Pairs of Monosomes Linked by IgG: Poly- dimer peak, as well as some nondigested disomes. The peak at somes (4 A~o units) were incubated with 300/~g of IgO at 0°C for 40 min. Then, the top of the gradients contains the RNase and unreacted IgG-reacted polysomes were incubated at 0°C for 30 min with 40/tg of RNase IgG's. The broadened peaks in Fig. 2A result from overlapping A (Sigma Chemical Co., St. Louis, MO) to cleave the message. Pairs containing of the cytoplasmic ribosomal peaks with those comprising of two monosomes linked by one IgG were separated from monosomes in a 15-30% chloroplast ribosomes which represent a significant proportion sucrose gradient in buffer A (E. coli) orb (L gibba) using a VTi 65 rotor (Beckman Instruments, Inc.) (I 13,000 g for 35 rain). The dimer peak was passed of the total ribosomes in the plant. through a Sepharose 6B column to remove sucrose. Electron micrographs of dimcrs of monosomes connected by Ribosomes were negatively stained with 1% uranyl acetate as described (21). IgG's are shown in Fig. 3. The most common views of IgG- Electron micrographs were obtained with a Philips 400 microscope at a magni- linked ribosomes correspond to the nonoverlap projection (10) fication of 64,500. in both eucaryotic (Fig. 3A) and procaryotic (Fig. 3 C) ribosomes. Also shown are monosomes attached to IgG's in their RESU LTS lower line of each figure. In the nonoverlap projection, the exit Both the plant L. gibba G-3 and E. coli mutant A324-5 produce site of the polypeptide nascent chain maps on the large subunit, large amounts of ribulose-l,5-bisphosphate carboxylase (18) ~70 A from the interface between subunits. and B-galactosidase (17), respectively. This allowed us to work with polysomes relatively enriched in these proteins. Typical DISCUSSION profdes of polysomes from L. gibba and E. coli are shown in It has been appreciated for some time that, on a gross scale, Fig. 1 A and B, respectively. E. coli polysomes have a maximum eucaryotic and procaryotic ribosomes share common structural of Ae56 at approximately 10 ribosomes per message. L. gibba organization (1). More recent comparative studies show that polysomes, in contrast to E. coli polysomes, contain two classes ribosomes from all three lineages, archaebacteria, eubacteria, of ribosomes, cytoplasmic (80S) and chloroplast (70S). and eucaryotes, share many common structural features (2, 16) Polysomes were reacted with IgG's against their respective such as the platform and cleft of the small subunit and the nascent protein chains. The specificity of these IgG's has been central protuberance and L7/LI2 stalk of the large subunit. 1472 R,PIpCOMMUNiCATiONS FIGURE 3 Electron micrographs of ribosomes reacted with IgG's against their nascent protein chains. In A and B eucaryotic, ribosomes are reacted with IgG's directed against Rub- isco and in C and D procaryotic ribosomes are reacted with IgG's directed against j~-galactosidase. Pairs of ribosomes (the honorer- lap projection) linked by an IgG are shown in A and C and single ribosomes with an attached IgG are shown in B and D.

Other ribosomal features, such as the archaebacterial bill and may traverse the ribosome in the fully extended conformation. the eucaryotic lobes, are present in only some lineages and The nascent chain exist site is located in an area where a absent in others (16). Less is known about the functional membrane binding site has been reported in lizard oocytes correspondence although it has been generally assumed that (25). The exit site and the membrane binding site probably parts of the ribosomes directly involved in translation (e.g., correspond to the two types of interactions in the attachment tRNA binding site, elongation factor binding sites, mRNA of eucaryotic ribosomes to membranes of the RER. One of binding site) are similar. No information exists about the these is through the nascent chain and the second possibly ribosomal locations of those functions involved in protein involves integral membrane proteins (26-28) and a signal secretion and transport, where ribosomal function might pos- recognition particle (29). Together, these two sites comprise the sibly differ in procaryotes and eucaryotes. The results in this region involved in the secretion of the proteins that we have paper provide the first information on the sites of these func- named exit domain (19). When ribosomes are bound with the tions. exit site contacting the membrane, the parts of the ribosome The nascent polypeptide chain exists from a similar ribo- involved in translation, i.e., the translation domain, faces the somal site in both procaryotic and eucaryotic ribosomes. In the cytoplasm. This is consistent with the requirement that the 60S subunit of L. gibba the nascent polypeptide chain emerges translational surface of the ribosome has access to ligands in 160 A from the central protuberance. In E. coil 50S subunits, the cytoplasm such as mRNAs, tRNAs, and factors. the nascent chain exists 140 A from the central protuberance, In conclusion, immune electron microscopy has shown that the site of the peptidyl transferase center (11). The conclusion the exit site of the nascent chain is located at similar regions in that the nascent chain exit site is quite distant from the peptidyl both procaryotes and eucaryotes and, in combination with transferase center is consistent with protease protection exper- other results, delineates the exit domain of the eucaryotic iments on the nascent polypeptide chain. These experiments ribosome. It is hoped that these observations will be useful in showed that the first 30--40 residues at the carboxy-terminus of ultimately understanding the molecular mechanisms of protein the nascent chain are protected from degradation, e.g., in synthesis and secretion. Bacillus subtilis (22), rabbit reticulocytes (28), and rat liver (24). From the distance between the exit site of the nascent chain We thank J. Beyer for excellent electron microscopy and J. Washizaki and the peptidyl transferase determined in our mapping exper- for photography. C. Bernabeu is a recipient of a Public Health Service International iments (and from the uncertainties in ribosomal dimensions Research Fellowship (5 F05 TWO 2747). Supported by grants from [_+15%]), we calculated that 39 +_ 6 and 44 + 7 residues could the National Science Foundation (PCM 76-14718, and PCM 78-19974) be protected in procaryotes and eucaryotes, respectively, if we to J. A. Lake and I. Zabin, respectively, and from the National Institute assume that the nascent chain is fully extended. Hence the of General Medical Sciences (GM 24034, GM 23167, and AI-04181) nonextended protein conformations, for example the alpha to J. A. Lake, E. Tobin, and I. Zabin, respectively. helix which measures 1.5 A/residue, would require many more residues to span this distance and are not consistent with the Received for publication 21 December 1982, and in revised form 19 protection measurements. This suggests that the nascent chain January 1983. RA~I~ COMMUNICATIONS 1473 REFERENCES Topography of RNA in the ribosome: location of the Y-end of 5S R.NA on the central protuberance of the 505 subunit. FEBS (Fed. Eur. Biocham. Soc.) Lett. 121:97-100. 1. Lake, J. A., Y. Nonomura, and D. D. Sahatini. 1974. Ribosome structure as studied by 14. g~ycharz, W. A., M. Nomura, and J. A. Lake. 1978. Ribosomal proteins L7/LI2 localized electron microscopy." In Ribosomes. M. Nomura, A. Tissieres, and P. 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